Collagenase synthesis in clonal monkey kidney epithelial cells (JTC-12 cells)

Cells of clone JTC-12 derived from monkey kidney epithelial cells were found to produce latent collagenase in serum-containing cultures. Collagenase production by JTC-12 cells was stimulated more than ten-fold with the addition of 12-0-tetradecanoyl-phorbol-13-acetate. The JTC-12 cell collagenase was purified 2,370-fold over the conditioned medium to a specific activity of 5,940 units/mg with a yield of 79% by heparin-Sepharose, ion-exchange and immunoadsorption chromatography. Thus the JTC-12 cell culture appears to be a useful model for studying the initial degradation of surrounding connective tissues by epithelial tissue in carcinogenesis.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Characterization of Na+-dependent phosphate transport in cardiac sarcolemmal vesicles

Pi uptake by purified bovine cardiac sarcolemmal vesicles was stimulated by an inwardly directed Na+ gradient, but not by such gradients of K+, Rb+, Li+, and choline. When Na+ was present both inside and outside the vesicles, or when Na+ gradient was dissipated by monensin, the Na+-dependent Pi uptake increased with time, reached a peak, and then declined approaching a steady state. The initial rate of Na+-dependent Pi uptake was a saturable function of Pi concentration (Km = 0.5 mM). These findings indicate the existence of a Na+,Pi-cotransporter in the sarcolemma. The Na+-activation curve of the Pi uptake exhibited positive cooperativity, suggesting the requirement for multiple Na+ binding to the functional unit of the carrier. The initial rate of Na+-dependent Pi uptake decreased as extra-vesicular pH increased in the range of 5.5-8.7. The uptake rate increased under conditions that are known or expected to generate an inside-negative membrane potential, indicating that Pi uptake is accompanied by the uptake of positive charge. These results suggest the electrogenic cotransports of two Na+ and one H2PO4-. We conclude that this cotransporter catalyzes the secondary active transport of Pi across the cardiac plasma membrane and regulates myocardial energy metabolism. We also suggest that the cotransporter may control intracellular Na+ and thus be involved in the regulation of trans-sarcolemmal Ca2+ movement and cardiac contractility.     

Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney

Summary A Na⁺-dependent hexose transport system with similar characteristics to that observed in the kidney is retained in a cultured epithelial cell line from pig kidney (LLC-PK₁). The active transport of methyl-d-glucoside ( MGP), a nonmetabolizable sugar, which shares the glucose-galactose transport system in kidney cells is mediated through a Na⁺-dependent, substrate-saturable process. The kinetic analysis of the effect of Na⁺ on the uptake of MGP indicated that the Na⁺-sugar cotransport system is an affinity type system in which the binding of either sugar or Na⁺ to carrier increases the affinity for the other ligand without affecting theV _max. The sequence of selectivity for different sugars studied by the inhibition produced in the uptake of MGP is very similar to that reported in rat kidney, rabbit kidney cortex slices, and rabbit renal brush border membrane vesicles. Phlorizin, even at very low concentration, almost completely inhibits MGP uptake. Conversely, phloretin at the same low concentration stimulated the sugar accumulation by inhibition of efflux, probably at the level of the basolateral membrane. Sulfhydryl group inhibitors also blocked the MGP uptake, suggesting that these groups were required for normal functioning of the sugar carrier system. This sugar transport system is an important functional marker to study the molecular events associated with the development of polarization in epithelial cells.     

Differentiated properties characteristic of renal proximal epithelium in a cell line derived from a normal monkey kidney (JTC-12)

Summary The JTC-12 cell, an established cell line derived from a normal monkey kidney, was studied in an attempt to characterize the epithelial qualities. Phase contrast microscopy showed dome formation in confluent monolayers and electron microscopic examinations revealed the presence of numerous microvilli on the apical membranes and desmosome between cells. Sonicated cells showed activities of γ-glutamyl transpeptidase, leucine aminopeptidase, alkaline phosphatase, and trehalase, marker enzymes of renal proximal epithelium. Alkaline phosphatase activity exhibited the characteristics of a renal type isozyme. Furthermore, confluent JTC-12 monolayers exhibited Na⁺-dependent transport of hexose, amino acid as well as inorganic phosphate. These findings indicate that JTC-12 cells in monolayer culture maintain ultrastructural, biochemical, and physiological properties of renal proximal epithelial cells. This cell line will be useful for further studies on cellular functions of renal proximal epithelium. This work was supported in part by grants from The Ministry of Health and Welfare of Japan and from the Ministry of Education, Science and Culture of Japan.     

Na(+)-dependent high-affinity uptake of L-glutamate in cultured fibroblasts.

Uptake of 1 microM [3H]L-glutamate by cultured 3T3 fibroblasts was strongly dependent on extracellular Na+; it was reduced by elevated concentrations of K+ (60 mM) but it was not influenced by variations in the concentration of Ca2+ (0-9.6 mM). D- and L-Asparate, D- and L-threo-3-hydroxyaspartate DL-threo-3-methylaspartate and a few other glutamate derivatives and analogues inhibited the uptake but several close analogues of L-glutamate (including D-glutamate) had no effect, implying that the uptake system is highly structurally selective. The recently identified inhibitor of glutamate uptake in synaptosomal preparations, L-trans-pyrrolidine-2,4-dicarboxylate, was also among the inhibitors. Apparent Km of the uptake was found to be less than 10 microM. The present observations indicate that Na(+)-dependent 'high-affinity' uptake of L-glutamate may appear in structures which are apparently unrelated to glutamatergic synaptic transmission in the CNS.     

Characterization of neutral amino acid uptake by cultured epithelial cells from pig kidney

Two transport systems for neutral amino acids have been characterised in LLC-PK₁ cells. The first, which transport alanine in a sodium-dependent manner, also mediates alanine exchange and is preferentially inhibited by serine, cysteine, and α-amino-n-butyric acid. This system resembles the ASC system in Ehrlich ascites and some other cell types. There is only a small contribution of other systems to alanine uptake. The second, which transports leucine with no requirement for sodium and mediates leucine exchange, is blocked by 2-aminonorbornane-2-carboxylic acid and hydrophobic amino acids. This system is similar to the L system described in other cell types. LLC-PK₁ cells retain several other features implying renal proximal tubule origin; our results thus suggest that these transport systems may be involved in the reabsorption of neutral amino acids by the nephron in vivo.     

Uptake of 22Na+ by cultured dog kidney cells (MDCK).

22Na+ uptake measurements were conducted on the dog kidney cell line, MDCK, to determine the mechanism of ouabain-insensitive sodium transport. The radioisotope was found to be taken up into monolayer cultures via an ATP-independent, saturable process (Km = 40 mM). The presence of sodium on the opposite side of the membrane gave rise to a transstimulation of the 22Na+ flux. Studies utilizing potassium and valinomycin suggested that the transport system was insensitive to changes in the membrane potential. Replacement of chloride in the assay buffer with other anions did not decrease the rate of 22Na+ uptake at 14 mMNa+, but bicarbonate and acetate were stimulatory. Potassium and rubidium increased the rate of 22Na+ influx (Ka = 13mM with 14 mM NaCL in the medium). Lithium (Ki = 7.5mM) and amiloride (Ki = 1.7 x 10(-5) M) were competitive and partially (or mixed) competitive inhibitors, respectively. The data are consistent with a mechanism of sodium uptake that includes a carrier(s) capable of catalyzing net sodium uptake and sodium-sodium exchange.     

Na+/K+-dependent adenosinetriphosphatase activity in mammalian kidney.

Microsomal preparations with Na+/K+-dependent ATPase activity from the outer medulla of rabbit and pig kidney were obtained. Purifications were assaied by centrifugation on sucrose discontinuous gradients and gel-filtration on Sepharose 6B, after detergent incubation. Sodium dodecylsulfate, with ATP as protecting agent, can remove a maximal amount of non specific proteins from the membranes and allows the recovery of a fraction with very high specific activity. The approach to purification by affinity chromatography techniques leads to interesting results, which induce us to pursue the present researchs to applicate an affinity method to Na+/K+-ATPase.     

Characterization of a Ca-dependent maxi K channel in the apical membrane of a cultured renal epithelium (JTC-12.P3)

Summary A Ca and potential-dependent K channel of large unit conductance was detected in the apical membrane of JTC-12.P3 cells, a continuous epithelial cell line of renal origin. The open probability of the channel is dependent on membrane potential and cytoplasmic-free Ca concentration. At cell-free configuration of the membrane patch, the open probability shows a bell-shaped behavior as function of membrane potential, which decreases at larger depolarization. With increasing Ca concentration, the width of the bell-shaped curve increases and the maximum shifts into the hyperpolarizing direction. For the first time the kinetics of this channel was analyzed under cell-attached conditions. In this case the kinetics could sufficiently be described by a simple open-closed behavior. The channel has an extremely small open probability at resting potential, which increases exponentially with depolarization. The low probability induces an uncertainty about the actual number of channels in the membrane patch. The number of channels is estimated by kinetic analysis. It is discussed that this K channel is essential for the repolarization of the membrane potential during electrogenic sodium-solute cotransport across the apical membrane.     

Ouabain-insensitivity of highly active Na+, K+-dependent adenosinetriphosphatase from rat kidney.

Highly purified preparations of Na⁺+K⁺-dependent adenosinetriphosphatase were isolated from rat kidney by two different procedures. The I₅₀ values for ouabain inhibition of the rat kidney enzyme at various stages of purification were determined to be essentially the same for all fractions tested (0.7 to 1.0 × 10^?4M). These results suggest that the marked insensitivity of the rat enzyme to inhibition by cardiac glycosides is due to the primary structure of the enzyme, and not to some other component in the tissue.     

Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018+/-0.009 in a Na+-free medium to 0.73+/-0.3 mM.l cells-1.min-1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e=24 mM; maximal velocity=11 mM.l cells-1.min-1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375+/-105 microM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.     

Inhibition of human intestinal brush border membrane vesicle Na+-dependent phosphate uptake by phosphophloretin derivatives

Hyperphosphatemia and II(o) hyperparathyroidism are common and severe complications of chronic renal failure. Reduced dietary phosphorus has been shown to be an effective treatment in reducing serum phosphate and serum PTH. 2(')-Phosphophloretin inhibited small intestine apical membrane Na(+)/phosphate cotransport and reduced serum phosphate in adult rats. 2(')-PP and phosphoesters of phloretin were tested for inhibition of human small intestine brush border membrane alkaline phosphatase activity and for inhibition of Na(+)-dependent phosphate uptake. The IC(50)'s for inhibition of alkaline phosphatase suggested an order of inhibitory potency of 4-PP > phloretin > 4(')-PP > 2(')-PP. Inhibition of Na(+)-dependent phosphate uptake followed the sequence 2(')-PPz.Gt;4(')-PP > 4-PP > phloretin. These results are consistent with 2(')-PP being a specific inhibitor of human intestinal brush border membrane Na(+)/phosphate cotransport.